Description (Adapted from applicant's abstract: Staphylococcus aureus is a major cause of human disease, especially nosocomial infections. It is also the third most common cause of confirmed bacterial food borne disease in the United States. The organism produces one or more serologically distinct enterotoxins when growing in food and the ingestion of the preformed toxin is responsible for the vomiting and diarrhea symptomology which is the hallmark of staphylococcal food poisoning. Despite its usual association with food poisoning, the enterotoxins are also virulence factors for the bacterium. The toxins, by virtue of being superantigens, can elicit a polyclonal T-cell activation in an infected individual. This activation diminishes the capacity of the individual to mount an appropriate immune response against the bacterial infection. Expression of many of the enterotoxins, like that of other virulence-associated exotoxins of S. aureus, is enhanced when activated by the accessory gene regulator (agr) network. The agr system involves a two component regulatory system which functions as a quorum sensor in S. aureus. It is thought that this system maximizes exotoxin production at a time in the infectious process when the host is mounting an inflammatory-response to the infection and the organism must respond to fight off the phagocytic cells. Consistent with this are the findings that agr mutants, which cannot activate exotoxin production, are significantly less virulent than then wild-type parent strain. This project utilizes the enterotoxin B and D genes as a model system to determine how the agr system works to activate exotoxin expression. Short DNA fragments from the promoter region of these enterotoxin genes have been positioned in front of a chloramphenicol acetyltransferase reporter gene and have been shown to contain the sequences necessary for agr related activation of expression. In this project, site-specific mutations will be introduced into the sequence and the specific bases responsible for the agr activation will be identified. The nature of the regulatory species responsible for the enhanced expression will be identified. The molecular nature of the interaction between the effector species and the enterotoxin gene promoter will be defined. The specific role of RNAIII, the effector species first generated by the initial activation of the two component system, will be evaluated with regard to enhancement of enterotoxin gene expression.